The Difference of Clinical Characteristics Between Patients With Exon ...

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OBSERVATIONAL STUDY

The Difference of Clinical Characteristics Between Patients With Exon 19 Deletion and Those With L858R Mutation in Nonsmall Cell Lung Cancer Yaxiong Zhang, MD, Dacheng He, MD, Wenfeng Fang, MD,PhD, Shiyang Kang, MD, Gang Chen, MD, Shaodong Hong, MD, Jin Sheng, MD, Jianhua Zhan, MD, Nan Chen, MD, Zhihuang Hu, MD, Cong Xue, MD, Yunpeng Yang, MD, Yuxiang Ma, MD, Tao Qin, MD, Ting Zhou, MD, Yan Huang, MD, and Li Zhang, MD

Abstract: Recent studies have demonstrated that exon 19 deletion (19 Del) and exon 21 L858R mutation (L858R) are 2 different types of sensitive epidermal growth factor receptor (EGFR) mutations in nonsmall cell lung cancer (NSCLC). However, whether there are some differences between those 2 groups in baseline clinical characteristics is still unclear. We enrolled consecutive 1271 NSCLC patients detected with either 19 Del or L858R and collected their baseline clinical characteristics including age, sex, comorbidity, smoking and drinking status, body mass index (BMI), TNM stage, histologic type, differentiation, tumor maximum diameter (TMD), and CEA level. x2 test and multivariate logistic regression analysis were used to compare the difference. We found a higher percentage of 19 Del in younger patients group (< ¼ 50 yr) than L858R (P < 0.001) through x2 test. Besides, patients with 19 Del have higher risk of lymph node metastasis (P < 0.001). However, there were no significant differences in other items of clinical characteristics between 19 Del and L858R. Multivariate analysis showed similar significant results. Subgroup

Editor: Yinyuan Wu. Received: August 1, 2015; revised: October 6, 2015; accepted: October 7, 2015. From the Sun Yat-sen University Cancer Center, State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine (YZ, DH, WF, SK, GC, SH, JS, JZ, NC, ZH, CX, YY, YM, TQ, TZ, YH, LZ); and Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China (YZ, DH, SK, GC). Correspondence: Li Zhang, Department of Medical Oncology, Sun Yat-sen University Cancer Center, 651 Dongfeng Road East, Guangzhou, Guangdong 510060, P.R. China (e-mail: [email protected]). YZ, DH, and WF contributed equally to this work. All the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the article. This study was supported by the following funds: (1) National High Technology Research and Development Program of China (Grant No. 2012AA02A502); (2) Innovative drug R&D center based on real-time high-throughput cell-based screening platform and large capacity compound library (Grant No. 2013ZX09401003-002); (3) National Natural Science Funds of China (Grant No. 81372502); (4) Natural Science Foundation of Guangdong (Grant No. S2013010016564); (5) Specialized Research Fund for the Doctoral Program of Higher Education (20120171120116); (6) Young Teacher Training Program of Sun YatSen University (14ykpy38); (7) Outstanding Young Talent Cultivation Project of Sun Yat-Sen University Cancer Center (04140701); (8) Wu Jieping Medical Foundation Project (Grant No. 320.6750.131). The authors have no conflicts of interest to disclose. Copyright # 2015 Wolters Kluwer Health, Inc. All rights reserved. This is an open access article distributed under the Creative Commons Attribution-NoDerivatives License 4.0, which allows for redistribution, commercial and non-commercial, as long as it is passed along unchanged and in whole, with credit to the author. ISSN: 0025-7974 DOI: 10.1097/MD.0000000000001949

Medicine

Volume 94, Number 44, November 2015

analysis in different age groups (10 yr as an interval) and N stages (stratified by N0, N1, N2, and N3) also indicated above-mentioned trends. NSCLC patients with 19 Del are more likely to be young and have lymphatic metastasis than those with L858R. Age and N stage might be considered in predicting EGFR mutation type in NSCLC. (Medicine 94(44):e1949) Abbreviations: 19 Del = exon 19 deletion, ALK = anaplastic lymphoma kinase, BMI = body mass index, CEA = carcinoembryonic antigen, EGFR = epidermal growth factor receptor, EGFR-TKI = EGFR tyrosine kinase inhibitor, KRAS = kirsten rat sarcoma, L858R = exon 21 L858R mutation, NSCLC = nonsmallcell lung cancer, OR = odd ratio, TMD = tumor maximum diameter.

INTRODUCTION

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onsmall-cell lung cancer (NSCLC) is the predominant form of lung cancer, the leading cause of cancer-related mortality worldwide, and patients are usually diagnosed in the advanced stages of disease.1 –4 NSCLC could be caused by the accumulation of genetic alterations. The most common one is kirsten rat sarcoma viral oncogene (KRAS) mutations (22%), followed by epidermal growth factor receptor (EGFR) mutations (17%), and anaplastic lymphoma kinase (ALK) rearrangement (7%).5 EGFR mutations can be divided into common EGFR mutations (19 Del/ L858R) and rare EGFR mutations. While common EGFR mutations considering EGFR tyrosine kinase inhibitors (EGFR-TKIs) as first-line treatment,6 platinum-based chemotherapy should be a first-line treatment for rare EGFR mutation.7 –9 However, 19 Del and L858R are 2 different types of sensitive EGFR mutations in NSCLC. Recent studies have reported that 19 Del and L858R have different responses to EGFR-TKIs. EGFR-TKIs treatment is more effective than chemotherapy in 19 Del patients. But for the patients with L858R, EGFR-TKIs treatment and chemotherapy have similar effect and chemotherapy might even be better.10,11 This breaks the previous idea that EGFR mutation patients should use TKI as possible. It also indicated that the 2 population, 19 Del patients and L858R ones, are different. However, whether there were any differences between those 2 groups in clinical characteristics is still unclear. Therefore, we sought to conduct a retrospective study to assess the difference of clinical characteristics between patients with 19 Del and those with L858R in nonsmall cell lung cancer in southern Chinese. www.md-journal.com |

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FIGURE 1. Flow chart of the enrollment.

MATERIALS AND METHODS Study Population We identified 1323 records of patients who had EGFR gene mutation positive in 5248 patients from October 2008 to April 2014 at Sun Yat-Sen University Cancer Center, Guangzhou, China. The study was approved by the Institutional Review Board of Sun Yat-Sen University Cancer Center. All the patients had provided written informed consent before samples were collected. In these records, 5 are neither 19 Del nor L858R. Two records are missing too much information while another 5 are repeat records. Therefore, we included 1311 records of patients. Figure 1 summarizes the flow chart. Among these patients, 4 are mixed mutation of 19 Del and L858R. Two are small cell lung cancer patients and 25 of them are not lung cancer patients. In addition, 3 patients’ lung cancer is metastatic. Another 4 patients are cancer in situ of the lung with T-stage marking Tis. The above patients were excluded and finally 1271 patients were included in the study. The clinicopathological features of the patients included age, sex, and comorbidity, smoking history, drinking history, body mass index (BMI), TNM stage, histologic type, differentiation, tumor maximum diameter (TMD), and carcino-embryonic antigen (CEA) level. Categories of each characteristic were divided as follows: for age, patients more than 50 years old were considered the older group. Smoking, alcohol history, and comorbidities were noted as yes or no. BMI equal to or larger than 24 kg/m2 was considered overweight group and the rest as normal group. Histologic type is divided into adenocarcinoma and non-adenocarcinoma. Differentiation is considered high, moderate, and low. High CEA level was defined, if CEA level in serum

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was >5 ng/mL. Similarly, large tumor was defined when the TMD was >3 cm. For N stage, we considered N0 as ‘‘without lymphatic metastasis’’ group and N1, N2, N3 as ‘‘with lymphatic metastasis’’ group. Similarly, M0 was defined as nonmetastasis group and M1 as metastasis group.

EGFR Mutation Detection EGFR mutations were detected using PCR-based direct sequencing of exons 18–21. The method is briefly introduced as follows. First, genomic DNAwas extracted from tumors embedded in paraffin blocks or fresh frozen tumors. Then, use Hot Star Taq DNA polymerase (Qiagen Inc, Valencia, CA) to complete PCR amplification with a forward primer (50 -GGATCGGCCTCTTCATGC-30 ) and a reverse primer (50 -TAAAATTGATTCCAATGCCATCC-30 ). Sequencing was performed by ABI PRISM 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA) using Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp., Foster City, CA) directly on PCR products. Any in-frame deletions in exon 19 or point mutations in exon 21 (L858R substitutions), which confer sensitivity to EGFR-TKIs therapy, were considered EGFR mutant.

Statistical Analyses SPSS 16.0 software was used for the statistical analysis. Continuous variables were divided into different categories as mentioned above. All the cut-off values were obtained by X-tile software (Version 3.6.1, Yale University, New Haven, CT), taking clinical expertise into consideration. Further investigations of multivariable analyses were performed by Cox Copyright

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Clinical Difference Between 19 Deletion and L858R

regression for factors which were significantly associated in univariate survival analyses. Results were reported with odd ratio (OR), corresponding 95% confidence intervals (CI). A P value 50 yr) and younger group (< ¼ 50 yr) were 68.4% (432/632) and 31.6% (200/632) in 19 Del and 79.8% (510/639) and 20.2% (129/639) in L858R, respectively. Univariate analyses indicated that the higher percentage of 19 Del in younger patients group (< ¼ 50 yr) than L858R was significant (P < 0.001). Multivariate analysis also showed similar significant result (P ¼ 0.011) (Table 2) We also conducted a subgroup analysis in different age groups. Age group was classified as 21–30, 31–40, 41–50, 51– 60, 61–70, 71–80, and 81–90 yr (10 yr as an interval). Nineteen Del patients showed higher percentage than L858R patients in all age groups below 61 yr, including 21–30, 31–40, 41–50, 51–60 yr, while lower percentage in all age groups above 60 yr, including 61–70, 71–80, and 81–90 yr (Figure 2A). This indicated same trends we found in univariate and multivariate analyses.

Patients With 19 Del Have Higher Risk of Lymph Node Metastasis Than L858R We assessed the lymph node metastasis rate of 19 Del and L858R patients. Lymphatic metastasis rates of 19 Del and L858R were 46.8% (296/632) and 39.6% (253/639), respectively. In univariate analysis, significant difference was observed in distribution of ‘‘without lymphatic metastasis’’ group (N0) and ‘‘with lymphatic metastasis’’ group (N1, N2, and N3) of each mutation. Patients with 19 Del had higher risk of lymph node metastasis than L858R ones (P < 0.001). Multivariate analysis showed the same result (P ¼ 0.002) (Table 2). We also conducted subgroup analysis in N stages (stratified by N0, N1, N2, and N3). The result showed that the percentages of N1, N2, and N3 in 19 Del were 9.7%, 23.6%, and 13.6%, respectively. And in L858R those numbers were 6.6%, 20.3%, and 12.7%, respectively. In each N stage, 19 Del had a higher percentage than L858R, except N0 where the percentage of 19 Del was 19.9% and L858R 29.0% (Figure 2B).

No Significant Differences in Other Items of Clinical Characteristics Between 19 Del and L858R There was no statistical difference in T or M or TNM stages between 19 Del and L858R patients in univariate Copyright

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NSCLC Patients With 19 Del Are More Likely to Be Young and Have Lymphatic Metastasis Than Those With L858R NSCLC, as one of the leading causes of cancer-related mortality around the globe, could be caused by the accumulation of genetic alterations. EGFR mutations, usually 19 Del or L858R, count for the second common type of genetic alteration in NSCLC. EGFR tyrosine kinase inhibitors (EGFR-TKIs) were considered to be the first-line therapy for advanced NSCLC patients harboring EGFR 19 Del and L858R.12–15 Although 19 Del and L858R are both common-type EGFR mutations and under the same first-line therapy recommendation, recent studies have shown that 19 Del patients benefit more from EGFRTKI therapy than L858R patients.10,11,16 However, few studies reported the difference of clinical characteristics between 19 Del and L858R patients. Based on these investigations, we hypothesized that there are some differences between the clinical characteristics of 19 Del and L858R patients. In our study, NSCLC patients with 19 Del did show differences with those L858R patients. NSCLC patients with 19 Del were more likely to be young. Besides, they are more likely to have lymphatic metastasis than L858R patients. We confirmed our findings by multivariate analysis. Subgroup analysis was also performed. Multivariate analysis showed the same trends above, as well as further subgroup analysis. However, there was no statistically significant difference among other aspects we observed in this study. The difference between 19 Del and L858R may help explain, in some aspects, why 19 Del patients benefit more from EGFR-TKI therapy than L858R patients. The reasons, to some degree, might lay to the young age of 19 Del patients and the inhibition of lymphatic metastasis of EGFR-TKI. Nineteen Del patients are more likely to be younger than L858R patients. Therefore, their basic conditions are better than L858R patients which could help in better prognosis. In addition, EFGR-TKI might inhibit the lymphatic metastasis of NSCLC patients and the inhibition effect would be greater in 19 Del patients because they are more likely to have lymphatic metastasis. The reason why 19 Del patients tend to be younger and have lymphatic metastasis is worthy of further research. Therefore, we concluded that NSCLC patients with 19 Del are more likely to be young and have lymphatic metastasis than those with L858R.

Age and N Stage Might Be Considered in Predicting EGFR Mutation Type in NSCLC Although testing of the mutations in EGFR, KRAS, and ALK is the today’s standard of care,15,17 a recent study has reported that the detection rate of epidermal growth factor receptor (EGFR) mutation in NSCLC patients in China was only 9.6% because of the limited prevalence of testing technology and that EGFR-TKIs were used more frequently as salvage rather than upfront therapy.18 This indicated the www.md-journal.com |

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TABLE 1. Baseline Characteristics of Enrolled 1271 Nonsmall-Cell Lung Cancer Patients With Either Exon 19 Deletions or Exon 21 L858R Mutations Parameter Age, yr Available patients (N ¼ 1271) Mean Range Sex Male Female Comorbidity No Tuberculosis Hepatitis B Hepatitis C Diabetes Hypertension Coronary heart disease Other NA Smoking No Yes NA Drinking No Yes NA BMI, kg/m2 Available patients (N ¼ 944) Mean Range T-stage 1a 1b 1a/1b 2a 2b 2a/2b 3 4 NA N-stage 0 1 2 3 NA M-stage 0 1a 1b 1a/1b NA TNM-stage IA IB IIA IIB IIIA IIIB

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Exon 19 Deletion (N ¼ 632)

L858R Mutation (N ¼ 639)

632 55.98 57 (27–84)

639 59.53 56 (28–84)

288 344

291 348

0.992

374 1 24 19 0 86 7 14 107

354 1 23 26 2 93 11 19 110

1.000 0.967 0.233 0.237 0.425 0.295 0.314 0.593

386 136 110

402 127 110

0.444 0.790

442 80 110

452 75 112

0.618 0.977

466 23.029 19.57 (14.57–31.14)

478 23.0195 20.34 (12.77–33.11)

32 25 35 115 15 92 31 87 200

39 26 32 143 13 79 40 77 190

0.666 0.400 0.941 0.446 0.216 0.866 0.261 0.336

126 61 149 86 210

185 42 130 81 201

0.001 0.002 0.021 0.005

259 82 142 4 145

280 79 126 0 154

0.521 0.187 0.038 0.902

41 58 32 13 73 41

49 97 13 16 70 28

0.211 0.005 0.945 0.475 0.083

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P


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Parameter

Clinical Difference Between 19 Deletion and L858R

Exon 19 Deletion (N ¼ 632)

L858R Mutation (N ¼ 639)

P

226 148

204 160

0.227 0.677

480 12 1 15 7 111

490 14 1 9 6 119

0.714 1.000 0.219 0.772 0.675

63 182 226 161

73 193 206 167

0.659 0.223 0.588

254 2.939 14.5 (0.5–15.0)

283 2.84 7.6 (0.4–8.0)

388 77.4243 4839.8 (0.2–4840)

395 127.33 15262.626 (0.374–15263)

IV NA Histologic type Adenocarcinoma Large cell carcinoma Adenosquamous carcinoma Squamous cell carcinoma other NA Differentiation High Moderate Low NA TMD, cm Available patients (N ¼ 537) Mean Range CEA, ng/mL Available patients (N ¼ 783) Mean Range

BMI ¼ body mass index, CEA ¼ carcinoembryonic antigen, NA ¼ not available, TMD ¼ tumor maximum diameter.

TABLE 2. The Difference of Baseline Characteristics Between NonSmall-Cell Lung Cancer Patients With Exon 19 Deletions and Those With Exon 21 L858R Mutations in Univariate and Multivariate Logistic Regression Analysis

Multivariate analysis (N ¼ 853)

Univariate Analysis Parameter Age, yr >50 < ¼ 50 Sex Male Female Comorbidity Yes No Smoking Yes No Drinking Yes No BMI, kg/m2 > ¼ 24